High Resolution Analysis of Rare Copy Number Variants in Patients With

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OPEN High resolution analysis of rare copy number variants in patients with autism spectrum disorder from Received: 20 April 2017 Accepted: 4 September 2017 Taiwan Published: xx xx xxxx Chia-Hsiang Chen1,2, Hsin-I. Chen3, Wei-Hsien Chien4, Ling-Hui Li5, Yu-Yu Wu1, Yen-Nan Chiu3, Wen-Che Tsai3 & Susan Shur-Fen Gau 3,6,7

Rare genomic copy number variations (CNVs) (frequency <1%) contribute a part to the genetic underpinnings of autism spectrum disorders (ASD). The study aimed to understand the scope of rare CNV in Taiwanese patients with ASD. We conducted a genome-wide CNV screening of 335 ASD patients (299 males, 36 females) from Taiwan using Afymetrix Genome-Wide Human SNP Array 6.0 and compared the incidence of rare CNV with that of 1093 control subjects (525 males, 568 females). We found a signifcantly increased global burden of rare CNVs in the ASD group compared to the controls as a whole or when the rare CNVs were classifed by the size and types of CNV. Further analysis confrmed the presence of several rare CNVs at regions strongly associated with ASD as reported in the literature in our sample. Additionally, we detected several new private pathogenic CNVs in our samples and fve patients carrying two pathogenic CNVs. Our data indicate that rare genomic CNVs contribute a part to the genetic landscape of our ASD patients. These CNVs are highly heterogeneous, and the clinical interpretation of the pathogenic CNVs of ASD is not straightforward in consideration of the incomplete penetrance, varied expressivity, and individual genetic background.

Autism spectrum disorder (ASD) represents a group of childhood-onset neurodevelopmental disorders char- acterized by abnormal social interactions, impaired verbal and nonverbal communication, and the presence of restricted interests and repetitive behaviors with long-term persistence of core features and functional impair- ment1,2. Te prevalence of ASD is various across diferent regions with an increasing trend over the years3–5 and with male excess in a male-to-female ratio of approximately 5:16–8. In the USA, the prevalence of ASD increased in the past decade according to the report of Centers for Disease Control and Prevention of USA8. It was estimated that around 1 in 68 persons aged eight years in the USA in 2010 was afected with ASD8. However, the increasing trend of ASD prevalence was not observed in the UK9. Te estimated prevalence of ASD in Chinese population ranged from 2.8 to 29.5 per 10,000 according to a recent review that summarized the fndings in Chinese population from several areas3. Te prevalence of ASD in Taiwan is approximately 0.3% based on the analysis of national health insurance research dataset10 and 1% based on the most recent Taiwan’s national sur- vey of child and adolescent mental disorders11 with a male: female ratio of approximately 4: 110. Due to its high prevalence, long-term impairment resulting in a great impact on individuals, families, and society12,13 and strong evidence of genetic components in its etiology14, this severe developmental disorder has been prioritized for molecular genetic studies15. Te heritability estimate of ASD is greater than 90%, attesting that genetic factors play a major role in the pathogenesis of ASD16–18. However, the genetics of ASD is very complex. Several genome-wide association studies (GWAS) have identifed some common single nucleotide polymorphisms (SNPs) associated with the risk of

1Department of Psychiatry, Chang Gung Memorial Hospital-Linkou, Taoyuan, Taiwan. 2Department and Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan. 3Department of Psychiatry, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan. 4Department of Occupational Therapy, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan. 5Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. 6Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan. 7Graduate Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan. Correspondence and requests for materials should be addressed to S.S.-F.G. (email: [email protected])

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CNV size ASD (n = 335) Controls (n = 1093) CNV rate ASD/CON Likelihood Ratio Chi-square P* <100 Kb 333 394 0.99/0.36 109.68 <0.0001 100–400 Kb 88 43 0.26/0.04 101.02 <0.0001 Deletion >400 Kb 18 14 0.05/0.01 15.41 <0.0001 Total 439 451 1.31/0.41 163.23 <0.0001 <100 Kb 263 229 0.79/0.21 146.25 <0.0001 100–400 Kb 150 144 0.45/0.13 84.17 <0.0001 Duplication >400 Kb 55 19 0.16/0.02 80.21 <0.0001 Total 468 392 1.40/0.36 224.09 <0.0001 <100 Kb 596 623 1.78/0.57 188.03 <0.0001 100–400 Kb 238 187 0.71/0.17 153.20 <0.0001 Deletion and Duplication >400 Kb 73 33 0.22/0.03 89.69 <0.0001 Total 907 843 2.71/0.77 273.33 <0.0001

Table 1. Comparisons of rare autosomal CNVs in patients with autism spectrum disorders and control subjects. P < 0.005 was considered statistically signifcant as it was corrected by 12 using Bonferroni test.

ASD, such as common variants on 20p12.1, 5p14.1, 1p13.219–23. Tese common SNPs, however, have only small efects on autism ASD risk, and of note, few if any of these SNPs were replicated in diferent studies. Furthermore, accumulating evidence suggests that rare genetic and genomic mutations also contribute to the genetics of ASD24. Conventional cytogenetic studies of ASD have revealed a variety of rare chromosomal abnormalities associated with ASD25–28 indicating aberrant genomic rearrangements are part of the genetic mechanism of ASD. Notably, the recent advent of array-based comparative genomic hybridization (aCGH) technology has discovered various submicroscopic copy number variations (CNVs) of genomic DNA associated with ASD29,30, leading further support to the idea that ASD is a genomic disorder in a subset of the patients. Tese ASD-associated CNVs are usu- ally individually unique and of low frequency, but together they account for approximately 5–10% of idiopathic ASD29, hence, constituting a part of the genetic architecture of ASD22,31,32. Te discovery of genomic mutations in ASD-associated CNVs not only helps decipher the genetic complexity of ASD23, but also helps shed some light on the neurobiology and pathogenesis of ASD32–37. In our previous studies, we reported four pathogenic CNVs in certain ASD patients38,39, indicating that CNVs also play a role in the genetic architecture of ASD in our patients. To have a better understanding of the scope of rare genomic CNVs in our ASD patient population, we recruited a sample of more than 300 ASD patients and conducted a genome-wide CNV screening in this sample. Results Clinical characteristics. A total of 335 (95.7%) out of 350 cases and 1093 (98.4%) out of 1111 controls passed a series of quality control of CNV experiments. We investigated the ethnicity of cases and controls by performing principle component analysis (PCA) with SNP genotype data from all the participants of this study and the individuals included in HapMap study. Te results demonstrated that the cases and controls are clustered together with the Han Chinese (Supplementary Figure 1). Terefore, the ethnicity of the participants of this study was confrmed to be the Han Chinese. Further, all the CNV data were subjected to the burden analysis. Te patient group consisted of 299 boys and 36 girls with the mean age of 9.4 ± 4.0 years, while the control group consisted of 525 males and 568 females with the mean age of 68.1 ± 10.1 years. Te ADI-R (Autism Diagnostic Interview-Revised) interviews revealed that the 335 patients scored 20.43 ± 6.12 in the “qualitative abnormalities in reciprocal social interaction”, 14.75 ± 4.32 in the “qualitative abnormalities in communication, verbal”, 8.19 ± 3.33 in the “qualitative abnormalities in communication, nonverbal”, and 6.95 ± 2.47 in the “restricted, repetitive and stereotyped patterns of behaviors.” All the participants with ASD were noted to have had abnormal development at or before 36 months of age. Teir current average intelligence quotients (IQ) were 94.85 ± 22.55 (range, 40 to 148) for full-scale IQ, 96.74 ± 2.04 (range, 41 to 145) for performance IQ, and 95.08 ± 23.79 (range, 44 to 148) for verbal IQ. Among the 335 ASD patients, nine had been diagnosed with epilepsy (3.04%), four had been suspected of seizure (1.35%), and 19 had ever had a febrile convulsion (6.42%). Tese data are also provided in the Supplementary Table 1.

CNV fndings. Te rates of rare CNV (<1% in the patients) at autosomes and X-chromosome were exam- ined between the patient and control groups. CNV regions on autosomes were analyzed in all samples while CNV regions on sex chromosomes were analyzed in male samples only. We found a signifcant excess of the overall rate of rare CNV at autosomes in the ASD patients (2.71) compared with the control subjects (0.77). Te over-representation of rare autosomal CNV rate in ASD was still present when the rare CNVs were grouped into deletion and duplication or classifed according to the size as <100 kb, 100–400 kb, and >400 kb (Table 1). In the analysis of rare CNV at X-chromosome, we compared only the rate of rare CNV between male patients and male control subjects. A signifcant excess of the overall rate of rare CNV was observed in the male ASD patients (0.214) compared with the male control subjects (0.011). Te excess rate of rare CNV at X-chromosome was still present when the CNVs were stratifed into deletion/duplication, or diferent size groups (Table 2). We did not compare the rate of rare CNV at X-chromosome between female patients and female controls, because of the random inactivation of X-chromosome in the females. Additionally, the sample size of the female patients is

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CNV size ASD (n = 299) Controls (n = 525) CNV rate ASD/CON Likelihood Ratio Chi-square P* <100 Kb 10 1 0.033/0.002 14.29 0.0002 100–400 Kb 7 1 0.023/0.002 8.98 0.0027 Deletion >400 Kb 8 0 0.027/0 16.08 <0.0001 Total 25 2 0.084/0.004 37.06 <0.0001 <100 Kb 6 1 0.020/0.002 7.26 0.0070 100–400 Kb 27 3 0.090/0.006 36.64 <0.0001 Duplication >400 Kb 7 0 0.023/0 14.09 0.0002 Total 40 4 0.134/0.008 55.05 <0.0001 <100 Kb 16 2 0.054/0.002 21.23 <0.0001 100–400 Kb 34 4 0.114/0.008 44.93 <0.0001 Deletion and Duplication >400 Kb 15 0 0.050/0 29.94 <0.0001 Total 65 6 0.217/0.011 88.74 <0.0001

Table 2. Comparisons of rare X-chromosome CNVs in male patients with autism spectrum disorders and male controls. P < 0.005 was considered statistically signifcant as it was corrected by 12 using Bonferroni test.

No. ID Sex Locus Start End Size (kb) Type Gene(s) involved Controls (n = 1093) 1 U-1902 Male 7q31.2 116365096 116443274 78 Dup MET 0 2 U-1638 Male 7q35 145064742 145950454 886 Dup CNTNAP2 0 3 U-1067 Male 15q11.2-13.1 24782255 28709280 3927 Dup PWRN1 to MIR4509 (144 gens) 1 (Dup) 4 U-1807 Male 15q11.2 23641502 28560804 4919 Dup GOLGA6L2 to HERC2 (148 genes) 1 (Dup) 5 U-2158 Male 15q13.3 32458661 32857470 399 Del CHRNA7 6 (1 Dup, 5 Del) 6 U-2233 Male 16p11.2 29591757 30191895 600 Dup SMG1P2 to MAPK3 (29 genes) 0 7 U-1199 Male 22q11.21-11.22 21917141 22970127 1053 Del UBE2L3 to LL22NC03-63E9.3 (14 genes) 0 8 U-1994 Male 22q11.21 18781534 19006984 225 Dup LOC102725072 to DGCR9 (6 genes) 9 (Dup) 9 U-801 Male 22q11.21 18640300 21611337 2971 Dup USP18 to FAM230B (83 genes) 0 10 U-830 Male 22q11.21 19024794 21611337 2587 Del DGCR2,–FAM230B (70 genes) 0 11 U-1459 Male 22q13.33 51127905 51234443 107 Del SHANK3, ACR, RABL2B, RPL23AP82 0 12 U-1957 Male 22q13.33 51087264 51234443 147 Del SHANK3, ACR, RABL2B, RPL23AP82 0 13 U-2239 Male 22q13.32-q13.33 49388701 51188494 1800 Dup C22orf34 to ACR (40 genes) 0 14 U-1344 Male Xp22.31 5940647 6666470 726 Dup NLGN4X 0*

Table 3. CNVs at the “hot spots” identifed in this study. *Only male controls (n = 525) were screened for this CNV at X chromosome.

small (n = 36) compared to the female controls (n = 568), and the skewed proportion of female patients vs. female controls (0.11 vs. 0.52).

CNVs at “hot spots”. We compared the rare CNVs found in our patients with the selected genetic “hot spots” of ASD as reported in the paper entitled “Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions” from the practice guideline of the American College of Medical Genetics and Genomics40. Te “hot spots” was defned as CNVs that have an especially strong association with ASD according to this paper. We identifed a total of 14 patients who had pathogenic CNVs located at several of the “hot spots.” Te detailed information of these CNVs including the locations, sizes, and genes encompassed in the CNV region are listed in Table 3, while the clinical data of each patient are listed in the Supplementary Table 2.

Other rare pathogenic CNVs. Besides the detection of CNVs at the “hot spots” in our sample, we further identifed a total of 49 rare putative pathogenic CNVs according to the “American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants”41 in our sample. Te pathogenic CNV was defned as “documented as clinically signifcant in multiple peer-reviewed publications, even if penetrance and expressivity of the CNV are known to be variable. Tis category includes large CNVs, which may not be described in the medical literature at the size observed in the patient but which overlap a smaller interval with clearly established clinical signifcance”41. Tese putative pathogenic CNVs overlapped with the pathogenic CNVs reported in the Clinical Genome Resources CNVs and DECIPHER. Table 4 presents the detailed information of these CNVs including locations, types, origins, and genes encompassed. Te clinical data of each patient are provided in the Supplementary Table 3.

Two-hit CNVs. Five patients were found to have two diferent putative pathogenic CNVs simultaneously in this study. Patient U-2075 inherited the 4q amplifcation and the 5p deletion from his mother and father, respectively (Fig. 1A). Patient U-1753 had the 8q amplifcation and the 8p deletion transmitted from his mother

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and father, respectively (Fig. 1B). Patient U-1255 acquired the 10q amplifcation and 18p amplifcation from his mother and father, respectively (Fig. 1C). Patient U-1414 had the 8p amplifcation from his father and a de novo 9q duplication (Fig. 1D). Patient U-1999 had two de novo amplifcations at 17q25.3 simultaneously (Fig. 1E). All the parents of these fve patients were carefully assessed, and none of them had ASD based on the self-administered questionnaires and clinical evaluation by the corresponding author. Te detailed information of these CNVs including the locations, sizes, and genes encompassed by these CNVs are listed in Table 5, and the clinical data of each patient are provided in the Supplementary Table 4. Discussion In this study, we compared the frequencies of rare CNVs (<1%) between 335 patients with ASD and 1093 control subjects from Taiwan. We found a signifcantly higher frequency of global rare CNVs in patients with ASD compared to the control group. Te signifcantly higher frequencies of rare CNVs in the ASD group were still present when the CNVs were subdivided into diferent groups based on deletion/duplication or the sizes. Our data are compatible with several previous studies24,33,42. Pinto and colleagues conducted a genome-wide CNV analysis of 996 ASD individuals of European ancestry and 1,287 matched controls. Tey found a higher global burden of rare genic CNVs in ASD patients43. Te fndings were replicated by the same group in another genome-wide CNV analysis consisted of 2,446 families with ASD33. In our study, we did not limit our CNV analysis to genic CNVs only, as non-genic CNVs may have position efect to afect the expression of genes outside the CNV regions. Our fndings of increased global burden of rare CNVs in ASD indicate that genomic rearrangement is one of the genetic mechanisms of ASD. Some other studies reported increased burden of CNV in female patients. Jacquemont and colleagues reported that in a sample of 762 ASD families, they found a 3-fold increase in deleterious autosomal CNVs in female patients compared to male probands44. Desachy and colleagues recently reported that mothers of patients with autism had a higher deletion burden than control mothers in a matched case–control population. Also, to their surprise, they found a higher autosomal burden of large, rare CNVs in females in the population. Tey speculated that the increased rare CNV burden in females in general population might contribute to the decreased female fetal loss in the population, but the ASD-specifc maternal CNV burden may contribute to high sibling recur- rence45. In our study, we did not conduct the similar analysis because of the relatively small sample size of female patients. In our CNV analysis, we identifed 14 patients who had CNVs located at the ASD “hot spots” as reported in the “Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions”40. Our fndings support the strong association of the CNVs at “hot spots” with ASD in our patient population. Among these CNVs, CNVs located at 22q11.2, 22q13.3, and 15q11-13 are the most common in our sample. Besides the CNVs located at the “hot spots,” we also detected 49 rare (<1%) CNVs larger than 400 kb that overlapped with the pathogenic CNVs reported in the Clinical Genome Resources and DECIPHER. Tese CNVs met the criteria of “pathogenic” according to the “American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants”41. Some of these rare putative pathogenic CNVs were inherited, while some were de novo mutations. Te broad distributions of both “hot spots” CNVs and rare pathogenic CNVs detected in our patients suggest extremely high genetic heterogeneity of ASD in our patients. Some studies suggested that female patients have a higher burden of CNV than male patients33,44,45. But, in our study, all the patients who had CNVs at “hot spots” were male (Table 3), and there were only two female patients out of 49 who had other rare (<1%) pathogenic CNVs (Table 4). Te discrepancy might be due to the disproportion of female patients in this study (36 females vs. 299 males). In this study, fve patients were found to have the concomitant presence of two rare pathogenic CNVs in their genome. Te fnding is consistent with our previous report of a patient who had inherited two CNVs from his parents and supported the two-hit model of ASD39. Several studies also proposed the idea of two-hit and the multiple-hit models of ASD, suggesting that genetic underpinnings of ASD stem from combinatorial efects of mutations of oligogenic or multiple genes in diferent loci39,46–48. Leblond and colleagues reported three patients with deletions at SHANK2 gene locus. Also, these three patients had another inherited CNV at 15q11-13 that was associated with other psychiatric disorders46. Two patients carried a duplication of nicotinic receptor CHRNA7, and one patient had a deletion of the synaptic translation repressor CYFIP146. Stenberg and Webber conducted a pathway-association test of target genes regulated by fragile-X mental retardation protein (FMRP) in ASD patients; they found rigorous support for the multiple-hit genetic etiology of ASD47. In fact, emerging evidence suggests the presence of multiple pathogenic CNVs in psychiatric patients is not rare. Hu and colleagues recently reported a novel maternally inherited 8q24.3 and a rare paternally inherited 14q23.3 CNVs in a family with neurodevelopmental disorders49. Williams and colleagues conducted CNV analysis in patients with velo-cardio-facial syndrome (VCFS), regardless of having psychosis or not. Tey found a signifcantly higher proportion of second CNV hit in patients with psychosis, suggesting the two-hit hypothesis may be relevant to a proportion of VCFS patients with psychosis50. Rudd and colleagues found a slightly higher proportion of multiple conservative CNVs in schizophrenia patients compared to controls, indicating a potential role for a multiple-hit model in schizo- phrenia51. Hence, it is likely that the multiple-hit (including two-hit) might be a commonly important genetic mechanism associated with ASD. In this study, we reported 5 patients who had two putative pathogenic CNVs larger than 400 kb. We believe that if CNVs smaller than 400 Kb were included for analysis in the future, we might fnd more patients with two-hit and multiple-hit of CNVs. In the family study of these 5 patients with two-hit CNVs, we found that some of these putative pathogenic CNVs were inherited from their parents, and some were a de novo mutation. However, the parents who carried one putative pathogenic CNV did not manifest ASD symptoms afer careful clinical evaluation, suggesting the incomplete penetrance of these inherited pathogenic CNVs. Further, we searched for these CNVs in 1093 control subjects, and found none of these CNVs in the control group, except the duplication of 18p11.31-p11.2 (483 kb), which was found in 5 out of 1093 control subjects

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No. ID Sex Locus Start End Size (kb) Type Origin Genes involved Controls (n = 1093) 1 U-728 Male 1q31.1 189384373 190699301 1315 Del BRINP3, KINC01351, LOC440704 0 2 U-1340 Male 2p13.1-12 74857355 75324605 467 Dup Mother MIAP, SEMA4F, HK2, LINC01291, POLE4, TACR1, MIR5000 0 3 U-866 Male 2p16.1 58164223 59360403 1196 Dup Father VRK2, FANCL, LINC01122 0 4 U-985 Male 2q14.3 125532806 126965458 1433 Dup Mother CNTNAP5 0 5 U-2170 Male 2q14.3-21.1 129497534 131211699 1714 Del Mother LOC101927881, to CYP4F62P (18 genes) 0 6 U-1726 Female 2q37.3 238318241 243089444 4771 Del de novo COL6A3 to LOC728323 (77 genes). 0 7 U-925 Male 3p24.3 19283615 19844654 561 Dup Father KCNH8, MIR4791 0 8 U-480 Male 4p13 44009201 44586716 578 Dup Father LVCAT1, KCTD8 0 9 U-2075 Male 4q12-13.1 58189742 62734625 4545 Dup Mother LOC101928851 to ADGRL3 (6 genes) 0 10 U-1535 Male 4q31.22 146812877 148129943 1317 Del de novo ZNF827 to TTC29 (8 genes) 0 11 U-1385 Male 5p12-11 45288787 46334867 1046 Dup Father HCN1 0 12 U-2058 Male 5p15.33 1685594 2197203 512 Dup de novo MIR4277 to CTD-2194D22.4 (6 genes) 1 (Del) 13 U-215 Male 5q13.2 68867282 70391241 1524 Dup Father GTF2H2C to LOC647859 (14 genes) 0 14 U-2075 Male 5q32 144772150 146559069 1787 Del Father PRELID2 to PPP2R2B-IT1 (11 genes) 0 15 U-1428 Female 7p14.1 39137061 39545773 409 Dup Father POU6F2, POU6F2-AS1 0 16 U-754 Male 7p22.3 1203841 1638496 435 Dup Father LOC101927021 to PSMG3-AS1 (8 genes) 0 17 U-890 Male 7q22.3 104,676522 105099343 423 Dup Father KMT2E, SRPK2, PUS7 0 18 U-717 Male 7q31.31 119313222 120243311 930 Dup Mother LVCAT5, KCND2 0 19 U-2829 Male 7q32.1 127266882 127670004 403 Dup Mother SND1, SND1-IT1, LRRC4 0 20 U-1753 Male 8p22 13277834 13841792 564 Del Father DLC1, C8orf48, LOC102725080 0 21 U-1130 Male 8p23.2 2179516 3890012 1710 Dup Mother LOC101927815, CSMD1 0 22 U-1414 Male 8p23.3 562629 1159,817 597 Dup Mother ERICH1, ERICH1-AS1, LOC401442 0 23 U-1753 Male 8q21.11 75793077 76234219 441 Dup Mother CRISPLD1, CASC9 0 24 U-363 Male 8q24.23 136620080 138711817 2092 Del Father KHDRBS3, LOC101927915 0 25 U-1511 Male 9p21.3 22190562 22988892 798 Dup Father DMRTA1, LINC01239 0 26 U-1269 Male 9q32 116314918 117370538 1056 Del de novo RGS3 to ATP6V1G1 (11 genes), 0 27 U-1414 Male 9q33.2-33.3 125422424 125947085 525 Dup de novo OR1L1 to, STRBP (16 genes) 0 28 U-1924 Male 10p11.21 34670528 35328422 658 Dup Father PARD3, PARD3-AS1, CUL2 0 29 U-1255 Male 10q26.2 128138653 128597833 459 Dup Mother C10orf90, DOCK1 0 30 U-1230 Male 12p11.1 33752330 34532722 780 Dup Father ALG10 1 (Dup) 31 U-1691 Male 12p11.1 33752330 34,532,722 780 Dup Father ALG10 1 (Dup) 32 U-1967 Male 12q24.33 130579,093 131130277 551 Dup Mother FZD10-AS1, FZD10, PIWIL1, RIMBP2 0 33 U-1850 Male 15q13.3 33145711 33546098 400 Dup Mother FMN1, TMCO5B 0 34 U-2015 Male 16p13.3 5942659 7000800 1058 Dup Mother RBFOX1 0 35 U-1578 Male 17p13.3 833790 1516480 683 Del de novo NXN to SLC43A2 (13 genes) 0 36 U-212 Male 17p13.3 172683 577890 405 Dup Father RPH3Al to VPS53 (6 genes) 2 (Dup) 37 U-1999 Male 17q25.3 78951153 79505624 554 Dup de novo CHMP6 to FSCN2 (22 genes) 0 38 U-1999 Male 17q25.3 79619226 80178991 559 Dup de novo PDE6G to CCDC57 (34 genes) 0 18p11.31- 39 U-1255 Male 7079985 7563165 483 Dup Father LAMA1, LRRC30 5 (Dup) 11.23 40 U-1519 Male 18p11.32 543161 2240220 1697 Dup Mother CETN1 to LINC00470 (8 genes) 0 19q13.42- 41 U-1957 Male 55237234 59097842 3860 Dup de novo KIR3DL3 to CENPBD1P1 (160 genes) 0 13.43 20q13.32- 42 U-2131 Male 58022339 59007873 986 Dup Father PHACTR3 to MIR646 (11 genes) 0 13.33 43 U-1452 Male 22q11.1 16055171 17330096 1275 Dup Mother DUXAP8 to HSFY1P1 (14 genes) 0 44 U-2200 Male Xp22.31 6467902 8135645 1668 Del PUDP to MIR651 (7 genes) 0* 45 U-1626 Male Xp22.31 6659217 7975015 1316 Del PUDP to PNPLA4 (6 genes) 0* 46 U-273 Male Xq12-13.1 67443166 67900951 458 Dup OPHN1, YIPF6, STARD8 0* 47 U-1160 Male Xp11.21-11.1 57384047 58438177 1054 Dup PAAH2, ZXD8, NLRP2B, ZXDA 0* 48 U-728 Male Xq21.33 96705629 98004847 1299 Dup DIAPH2, DAIPH2-AS1 0* 49 U-919 Male Xp22.31 6455151 8145721 1691 Dup PUDP to VCX2 (7 genes) 0*

Table 4. Other rare pathogenic CNVs identifed in patients and control subjects in this study. *Only male controls (n = 525) were screened for CNVs at X chromosome.

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Figure 1. Te pedigrees of fve patients who carry two CNVs and the origins of these CNVs. Dup: duplication, Del: deletion.

(0.46%). Tese data provided further evidence to support that these CNVs may confer increased risk to ASD. In the future, we might be able to identify unafected carriers of high-risk CNVs if more data are accumulated. Te identifcation of pathogenic CNVs associated with ASD may help discover candidate genes of ASD. Te genes encompassed by the CNVs in our patients are listed in Tables 3,4 and 5. Notably, several genes had been reported to be associated with autism or the other major psychiatric disorders, such as TACR152, CNTNAP553,54, ADGRL355, ZNF82756, POU6F220, KMT2E57, KCND258, SND159, CNTNAP260,61, CSMD162,63, PARD364, HERC265, FMN166, and RBFOX167. Tese fndings not only provide further clues to indicate the highly genetic heterogeneity of ASD but also indicate the pleiotropic clinical efects of the mutation of these genes. Accumulating evidence showed that shared heritability and genetic mutations among diferent categories of psychiatric disorders seem to be regular rather than exceptional. A study of analyzing the genotype data from the Psychiatric Genomics Consortium (PGC) for cases and controls in schizophrenia, bipolar disorder, major depressive disorder, ASD and attention-defcit/hyperactivity disorder (ADHD) revealed moderate to high shared genetic etiology of these psychiatric disorders68. Li and colleagues recently reported that the prevalence of de novo mutations shared by four diferent categories of neuropsychiatric disorders: autism spectrum disorder, epileptic encephalopathy, intellectual disability, and schizophrenia, was signifcantly elevated69. Te present study has several limitations. First, to the best of our knowledge, our study has the largest sample size of Chinese population compared to the other studies70,71. However, the relatively limited sample size of this

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ID Locus Start Stop Size (kb) Type Genes contained Controls (n = 1093) PRELID2, GRXCR2, SH3RF2, PLAC8L1, LARS, RBM27, POU4F3, U-2075 5q32 144772150 146559,069 1787 Del 0 TCERG1, PPP2R2B U-2075 4q12-13.1 58189742 62734625 4545 Dup LPHN3 0 U-1753 8p22 13277834 13841792 564 Del DLC1 0 U-1753 8q21.11 75793077 76234219 441 Dup CRISPLD1 0 U-1255 18p11.31-p11.23 7079985 7563165 483 Dup LAMA1 5 (Dup) U-1255 10q26.2 128138653 128597833 459 Dup C10orf90, DOCK1 0 OR1L1, OR1L3, OR1L4, OR1L6, OR5C1, OR1K1, PDCL, RC3H2, ZBTB6, U-1414 9q33.2-q33.3 125422424 125947085 525 Dup 0 ZBTB26, RABGAP1, GPR21, NCRNA00287, MIR600, STRBP U-1414 8p23.3 562629 1159817 597 Dup ERICH1 0 CHMP6, FLJ90757, BAIAP2, AATK, LOC388428, AZI1, C170rf56, U-1999 17q25.3 78951153 79505624 554 Dup 0 SLC38A10, C17orf55, TMEM105, BAHCC1, ACTG1, FSCN2 PDE6G, C17orf90, CCDC137, ARL16, HGS, MRPL12, SLC25A10, GCGR, FAM195B, P4HB, ARHGDIA, THOC4, NANPC11, NPB, PCYT2, SIRT7, U-1999 17q25.3 79619226 80178991 560 Dup 0 MAFG, LOC92659, PYCR1, MYADML2, NOTUM, ASPSCR1, STRA13, LRRC45, PAC3, DCXR, RFNG, GPS1, DUS1LFASN, CCDC57

Table 5. Locations, sizes and types of CNVs in patients with two hits.

study is an apparent limitation to have a more comprehensive picture of CNVs in our patient population, especially in consideration of the high heterogeneity of CNVs associated with ASD. Second, in this study, we only searched for pathogenic CNVs larger than 400 kb because larger CNVs are more likely to be pathogenic72. We understand that small CNVs can be pathogenic. Hence, further analysis of the CNVs smaller than 400 kb will discover more ASD-associated CNVs in our patients. Tird, the phenotypical interpretation of pathogenic CNVs found in our study is not straightforward given the incomplete penetrance, varied expressivity and pleiotropic efects of pathogenic CNVs identifed in our sample. Also, we cannot exclude the interaction of these CNVs with the genetic background and other yet to be identifed genetic or genomic mutations in the afected patients. In conclusion, we found a signifcantly increased global burden of rare CNVs in our ASD patients compared to the control subjects, indicating that rare CNVs play a part in the genetic landscape of ASD in our population. Also, we identifed several pathogenic CNVs at “hot spots” and various private putative pathogenic CNVs in our patients, suggesting high genetic heterogeneity of ASD in our patients. Our study also supports that high-resolution oligonucleotide SNP array is a useful tool to uncover the genetic underpinnings of patients with ASD. In the future, we will continue to analyze our data with the size of CNV smaller than <400 kb, and we expect to fnd more pathogenic CNVs associated with ASD, more candidate genes of ASD, and more patients with double-hit of CNV. Tus, we will have a better understanding of the genetic architecture of ASD in our population. Materials and Methods Participants and Procedures. The study protocol was approved by the Research Ethics Committee at National Taiwan University Hospital (approval number: 9561709027), Taipei, Taiwan, and Chang Gung Memorial Hospital-Linkou (approval number: 93-6244), Taiwan, for the recruitment of the patients with ASD, and Academia Sinica (approval number, AS-IBMS-MREC-91-10), Taiwan for the control group. All the experiments and informed consent procedures were performed in accordance with relevant guidelines and regulations set by the research ethics committees of the three institutes.

Patient Participants with ASD. Te study was part of the molecular genetics study of patients with ASD who were Han Chinese residing in Taiwan. Te detailed recruitment and evaluation of the patients and their family members were described in our previous publication73. In brief, patients aged 3 to 17 years old and met the clinical diagnosis of autistic disorder as defned by the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV)74 were recruited from the Children’s Mental Health Center, National Taiwan University Hospital, Taipei, Taiwan and Department of Psychiatry, Chang-Gung Memorial Hospital, Kuei-Shan, Taiwan. Te clinical diagnoses were made by board-certifed child psychiatrists experienced in the assessment and intervention for ASD and were further confrmed by interviewing the parents using the Chinese version of the Autism Diagnostic Interview-Revised (ADI-R)75,76. Te ADI-R, translated into Mandarin by Gau and colleagues, was approved by Western Psychological Services in 2007 as the ADI-R in the Chinese language75,77. All these patient participants further received clinical evaluation according to the DSM-5 diagnostic criteria for ASD1, which revealed that all the 350 participants with DSM-IV autistic disorder met the diagnosis of DSM-5 ASD. Moreover, all these patient participants received intelligence tests. For ages of 3 to 7.5 years, the Wechsler Primary and Preschool Scale of Intelligence-Revised (WPPSI-R) was given; for ages of 6 to 16 years 11 months, the Wechsler Intelligence Scale for Children-3rd Edition (WISC-III) was given; for ages of 16 years and above, Wechsler Adult Intelligence Scale (WAIS) was given. Patients with known chromosomal abnormalities and associated medical conditions including fragile X syndrome and Rett’s disorder based on DNA testing or clinical assessments were not included during the recruitment process28,78. Also, probands with previously identifed chromosomal structural abnormality associated with autism or had any other major neurological or medical conditions were also excluded28. Te study protocol was approved by the Research Ethics Committee of National Taiwan University Hospital (approval number,

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9561709027) and Chang Gung Memorial Hospital (approval number, 93-6244), Taiwan. Written informed consents were obtained from the participants (if applicable, otherwise, child assent) and their parents afer the procedures were fully explained. Genomic DNA was prepared from peripheral blood of each participant using Gentra Puregene Blood kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

Healthy control subjects. Te control subjects (n = 1111) were chosen from the Han Chinese Cell and Genome Bank (HCCGB) in Taiwan who received physical check-up and questionnaire screening to ensure that they did not have any abnormal physical condition and mental illness79. Written informed consents were obtained from the participants afer the procedures were fully explained.

CNV analysis. We used Afymetrix Genome-Wide Human SNP Array 6.0 (Afymetrix, Santa Clara, CA, USA) for genome-wide CNV screening. Te SNP 6.0 array contains more than 1.8 million markers including more than 906,600 probes for SNPs and more than 946,000 probes for CNVs. Tese probes are evenly distrib- uted across the whole genome with a median distance between probes of ~0.7 kb. Te microarray experiment was conducted by the National Genotyping Center (Academia Sinica, Taipei, Taiwan) (http://ncgm.sinica.edu. tw/ncgm_02/index.html). Te hybridization intensities were captured by GeneChip Scanner 3000 (Afymetrix, Santa Clara, CA). CNVs were called using Afymetrix Genotyping Console sofware v.4.1 (Afymetrix, Santa Clara, CA). Te average call rate was 99.49 ± 0.29%, and all samples passed genotyping quality control (call rate >= 95%). Te gender was called based on the cn-probe-chrXY-ratio_gender method from Afymetrix Power Tools (Afymetrix, CA, USA). Samples with mismatched gender between computed gender and case information were excluded from analysis. Duplicated samples detected by Kinship analysis using P-Link sofware were also excluded. Twenty contiguous deletion or duplication probe signals were called in this study. CNV regions overlapped with centromeric regions (hg19, UCSC), antibody variable regions (PennCNV, http://www.openbioinfor- matics.org/penncnv/penncnv_faq.html#ig) and T-cell receptor loci (NCBI Gene, http://www.ncbi.nlm.nih.gov/ gene/) were fltered out. Te CNVs with the size equal or larger than 10 Kb and with the frequency of less than 1% in the patients were selected for analysis in this study. Copy number variations were considered to localize at the same locus if they overlapped by at least 80% of their length. Genes overlapped with the CNV regions were reported according to UCSC genes (NCBI37/hg19). Te ethnicity of cases and controls was assessed by performing principle component analysis (PCA) with SNP genotype data from all the participants of this study and the individuals included in HapMap study.

Burden assay. Both genic and non-genic CNVs were included for analysis. Likelihood Ratio Chi-square test was used to compare the diference of CNV rate between ASD and healthy controls with a pre-selected alpha value at P value less than 0.05. Bonferroni correction was used to adjust for the multiple testing. Tus, the signif- icance level of the p-value was set at 0.005.

Real-time quantitative PCR (RT-qPCR). RT-qPCR was used to validate the CNVs detected in this study and for a family study to identify their parental origin. RT-qPCR was performed using the SYBR-Green PCR reagents kit (Applied Biosystems, Forster City, California, USA), and the CNV was assessed using a relatively standard method in the laboratory38. Te experiment was implemented using the ABI StepOnePlus following the manufacturer’s protocol (Applied Biosystems, Forster City, California, USA). Te description of primer sequences, optimal annealing temperature, and the amplicon sizes is available upon request.

Pathogenic CNV evaluation. Te pathogenic CNVs were evaluated according to two practice guidelines from the American College of Genetics and Genomics. First, according to the “Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions”40, our CNVs results overlapped with those at the “hot spots” reported in this guideline are considered as pathogenic. Second, for rare CNVs outside the “hot spots,” we focused on the analysis of rare CNVs equal or larger than 400 kb for convenience’s sake. Although large CNVs are more likely to have clinical signifcance, we understand that small CNV can be pathogenic, and large CNV can be benign. CNVs overlapped with the pathogenic CNVs reported in the Clinical Genome Resources (https://www.clinicalgenome.org/), or DECIPHER (https://decipher.sanger.ac.uk/) were defned as pathogenic according to the “American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants”41. Te sizes of CNVs and genes encompassed by the CNVs were generated according to the gene annotation of the UCSC genome browser (GRCh37/hg 19) (http://genome.ucsc.edu/cgi-bin/hgGateway). References 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition 5th edn, (American Psychiatric Association 2013). 2. Lai, M. C., Lombardo, M. V. & Baron-Cohen, S. Autism. Lancet. 383, 896–910 (2014). 3. Feng, L. et al. Autism spectrum disorder in Chinese populations: a brief review. Asia Pac Psychiatry. 5, 54–60 (2013). 4. Isaksen, J., Diseth, T. H., Schjolberg, S. & Skjeldal, O. H. Autism spectrum disorders–are they really epidemic? Eur J Paediatr Neurol. 17, 327–333 (2013). 5. Kim, Y. S. et al. Prevalence of autism spectrum disorders in a total population sample. Am J Psychiatry 168, 904–912 (2011). 6. Ganz, M. L. Te lifetime distribution of the incremental societal costs of autism. Arch Pediatr Adolesc Med. 161, 343–349 (2007). 7. Fombonne, E. Epidemiological surveys of autism and other pervasive developmental disorders: an update. J Autism Dev Disord. 33, 365–382 (2003). 8. CDC. Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2010. MMWR Surveill Summ. 63, 1–21 (2014). 9. Taylor, B., Jick, H. & Maclaughlin, D. Prevalence and incidence rates of autism in the UK: time trend from 2004-2010 in children aged 8 years. BMJ Open. 3, e003219, https://doi.org/10.1136/bmjopen-2013-003219 (2013).

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Tis work was supported by grants from Ministry of Science and Technology (NSC96-3112-B-002-033, NSC97-3112-B-002-009, NSC98-3112-B-002-004, NSC 99-3112-B-002-036, MOST 103-2314-B-002-055-MY3), National Taiwan University (AIM for Top University Excellent Research Project: 10R81918-03, 101R892103, 102R892103, 103R892103), and National Health Research Institute (NHRI-EX104-10404PI, NHRI-EX105-10404PI), Taiwan. Author Contributions Chia-Hsiang Chen and Susan Shur-Fen Gau designed and supervised the study; Hsin-I Chen, and Wei-Hsien Chien conducted the experiments; Ling-Hui Li analyzed the results; Yu-Yu Wu, Yen-Nan Chiu, and Wen-Che Tsai helped with the evaluation and collection of the samples. All authors reviewed drafs and approved the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-12081-4. Competing Interests: Te authors declare that they have no competing interests. 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